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US7060689B2 - Methods and compositions for delivery and retention of active agents to lymph nodes - Google Patents

Methods and compositions for delivery and retention of active agents to lymph nodes Download PDF

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US7060689B2
US7060689B2 US10/044,650 US4465002A US7060689B2 US 7060689 B2 US7060689 B2 US 7060689B2 US 4465002 A US4465002 A US 4465002A US 7060689 B2 US7060689 B2 US 7060689B2
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avidin
ligand
biotin
liposome
lymph nodes
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US20020164648A1 (en
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Beth A. Goins
William T. Phillips
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University of Texas System
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6897—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies
    • A61K47/6898—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies using avidin- or biotin-conjugated antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A61K51/1217—Dispersions, suspensions, colloids, emulsions, e.g. perfluorinated emulsion, sols
    • A61K51/1234—Liposomes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T428/00—Stock material or miscellaneous articles
    • Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
    • Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]
    • Y10T428/2984—Microcapsule with fluid core [includes liposome]

Definitions

  • the present invention relates generally to delivery and retention of active agents at a targeted site using compositions comprising ligand and anti-ligand conjugates. More particularly, certain embodiments concern methods, compounds, compositions and kits useful for targeted delivery and retention of agents at specific lymph nodes by administration of a composition comprising a ligand-colloid moiety and a composition comprising an anti-ligand.
  • the ligand is one member of the biotin/avidin pair and the anti-ligand is the other member of the biotin/avidin pair.
  • Avidins are a family of proteins functionally defined by their high affinity for binding biotin. Avidins are small oligomeric proteins made up of four identical subunits, each with a single binding site for biotin. Avidins include proteins (1) present in the eggs of amphibians, reptiles, and birds, and known as avidin, and (2) produced by Streptomyces avidinii and known as streptavidin. Streptavidin is similar to avidin in its binding properties, but has lower non-specific tissue binding, and therefore often is used in place of avidin. However, the immunogenicity of streptavidin is a major drawback to its use. Paganelli et al., Eur J Nucl Med 24(3):350–351, 1997.
  • Biotin is a natural water-soluble vitamin found in every living cell. Several derivatives of biotin are commercially available. Due to the extremely high affinity of avidins for biotin, the biotin/avidin system has been used for targeting, detecting, and treating tumors. The tetravalency of avidin for biotin permits amplification when avidin binds to biotin.
  • biotin/avidin system has been used primarily in conjunction with antibodies targeting specific tissues or lesions, as described, for example, in Hnatowich et al., Nucl Med Biol 20:189–195, 1993; Ogihara-Umeda et al., Cancer Detection and Prevention 21(6):490–496, 1997; Lanza, U.S. Pat. No. 5,690,907; Goldenberg, U.S. Pat. Nos. 5,736,119 and 5,776,094; and Griffiths, U.S. Pat. No. 5,482,698, incorporated herein in their entirety by reference.
  • Antibodies directed against different determinants associated with pathologic or normal cell type or pathogenic organisms have been used to target tissues and lesions, as described in, for example, Griffiths, U.S. Pat. No. 5,482,698 and Goldenberg, U.S. Pat. No. 5,776,094.
  • the biotin/avidin system has been used as a means for delivering a detection agent to a site previously targeted by antibody and for clearing excess targeting antibody from the circulation in order to increase the target:background ratio.
  • a targeting antibody is conjugated with either avidin or biotin and then is injected into a subject, thus localizing the avidin or biotin at a site of interest. Thereafter, either biotin or avidin (depending on which was coupled to the targeting antibody) bearing an active agent is injected and is localized at the site of the primary antibody by binding to avidin or biotin respectively. Timing of the second injection after the first one is very critical.
  • a drawback of both of the above approaches for targeting tumors is that they require that a subject be available to undergo multiple procedures over a prolonged time, generally a few days.
  • a targeting compound comprising a targeting moiety such as antibody conjugated to one member of the biotin/avidin pair is administered to a subject.
  • a portion of the targeting compound distributes to the targeted site and is bound there. The significant portion of the targeting compound may be retained in the blood pool.
  • a compound comprising the member of the biotin/avidin system that is not conjugated to a targeting moiety is administered and binds with the biotin or avidin of the circulating targeting compound.
  • a detection or therapeutic agent is incorporated with either the targeting compound, or the subsequently injected avidin or biotin compound, or both, prior to administration.
  • the newly formed compound comprising targeting moiety-biotin-avidin-detection/therapeutic agent is removed from circulation by the reticuloendothelial system, thus clearing the compound from those portions of the body that are not targeted and lowering the background level of the compound.
  • This increases the target-to-background ratio and increases the ability to detect the targeted site
  • High background levels of a detection agent have long been recognized as a major impediment to achieving the high target:background ratios desirable for detection of lesions.
  • high background levels of therapeutic agents can lead to toxicity and restrict the therapeutic agents and dosages that can be safely used for therapy.
  • Liposomes have been described as potential agents for targeted delivery of diagnostic or therapeutic agents to a wide range of organ systems and diseases. Such targeting is due primarily to a physical feature of the liposome, such as size, charge, and lipid compound, and is not due to specific site-directed targeting. Phillips et al., Handbook of Targeted Delivery of Imaging Agents , CRC Press, 149–173, 1995, incorporated herein in its entirety by reference. Coupling macrophage-specific ligands to the surface of liposomes increases liposome drainage from an interstitial injection site and enhances their localization in regional lymph nodes. Moghimi et al., Prog Biophys Molec Biol 65:221–249, 1996, incorporated herein in its entirety by reference.
  • the lymphatic system plays important roles in transporting body fluids and particulate materials from the body's periphery to the thoracic duct, returning large proteins and lymphocytes to the blood circulation from the tissue fluid, and transporting the products of fat digestion in the gastrointestinal tract (chylomicrons) into the blood circulation.
  • the properties of the lymphatic system have been reviewed in detail by Yrissay et al., Lymphatics, lymph and the lymphomyeloid complex, Academic Press, London, 1970, incorporated herein in its entirety by reference.
  • lymphatic system also plays an important role in the spread of a variety of disease processes, as described, for example, by Papisov et al., Crit Rev Ther Drug Carrier Syst 13(1&2):57–84, 1996, incorporated herein in its entirety by reference. Lymphatic dissemination of disease processes allows the spread of disease to regional lymph nodes, and even further. For example, malignant cells can enter the lymphatic system and become captured by lymph nodes; the lymph nodes consequently serve as foci of residual metastatic cancer, with potential tumor recurrence even after treatment of the primary tumor. Weiss et al., Lymphatic System Metastases , Hall, Boston, 1988. The lymph can also be involved in the spread of tumors to other organs.
  • lymph nodes any material which transits from the interstitial space to the intralymphatic space will move to a series of lymph nodes, the regional lymph nodes, that drain the lymph toward the thoracic duct.
  • the first such lymph node encountered is the primary lymph node. Lymph from the primary lymph node will pass to subsequent lymph nodes, called secondary lymph nodes.
  • the sentinel lymph node is the first lymph node encountered by a metastasizing tumor cell after it has entered the lymphatic system.
  • the importance of the sentinel lymph node lies in the fact that metastasizing tumor cells are recognized by the immune system and stopped there. Many times, these tumor cells are destroyed by the immune cells located in the sentinel lymph node. However, tumor cells can survive, creating a foci of metastatic disease in the sentinel lymph node.
  • Locating the sentinel lymph node in a mammalian subject is not always easily accomplished. Lymph nodes tend to blend in with the rest of the body tissue. In addition, it is not readily apparent which of numerous lymph nodes identified drains a disease locus, such as a tumor bed. Furthermore, occasionally a disease locus can drain to more than one area, thus there can be multiple sentinel lymph nodes.
  • the present invention addresses the need to identify sentinel lymph nodes.
  • colloids such as liposomes
  • lymphatic tissue Interstitial administration of colloids such as liposomes results in accumulation of such colloids in lymphatic tissue, and is described, for example, in Oussoren et al., Biochim Biophys Acta 1328:261–272, 1997, incorporated herein in its entirety by reference.
  • Efficient accumulation of macromolecular carriers in lymph nodes after intralymphatic injection of macromolecules has also been described. See, for example, Papisov, Crit Rev Ther Drug Carrier Syst 13(1&2):57–84, 1996.
  • microcolloidal particles labeled with a radioisotope are administered interstitially proximal to the tumor site and scintigraphic scans or radio-guided probes are used to locate the site(s) of maximum radioactivity.
  • This method is described, for example, in Van der Veen et al., Br J Surg 81(12):1769–1770, 1994; Krag et al., Surg Oncol 2:335–339, 1993; Veronesi et al., Lancet 349(9069):1864–1867, 1997, all of which are incorporated herein in their entirety by reference.
  • vital blue dye is injected peri-tumor, as described, for example, in Morton et al., Surg Oncol Clin N Am 1:247–59, 1992 and Arch Surg 127(4):392–399, 1992, incorporated herein in their entirety by reference.
  • An intraoperative method for detecting sentinel lymph nodes using both radiolabeled colloid and vital blue dye has also been described, for example, in Cox et al., Ann Surg 227(5):645–653, 1998, incorporated herein in its entirety by reference.
  • radiolabeled colloid is injected around the periphery of a tumor site one to six hours prior to an operative procedure.
  • vital blue dye is injected peri-tumor.
  • the vital blue dye stains afferent lymphatic channels to aid in visual localization of the sentinel lymph node.
  • a hand-held gamma-detection probe Prior to skin incision, a hand-held gamma-detection probe is used to localize the sentinel lymph node. After incision, the gamma probe is used to guide localization of the sentinel lymph node. See, for example, Kotz, J Nucl Med 39(12):13N–21N, 1998; Krag et al., N Engl J Med 339(14):941–946, 1998; Reintgen, J Nucl Med 39(12):22N–36N, 1998, all of which are incorporated herein in their entirety by reference.
  • a targeting system for the delivery of diagnostic and therapeutic agents to the lymphatic system should have the following characteristics: (i) spread well from the injection site, (ii) provide good uptake in primary lymph node(s) if sentinel lymph node is desired, and (iii) provide good uptake in secondary regional lymph nodes if desired.
  • the present invention relates generally to delivering and retaining a desired agent at a targeted site using a ligand/anti-ligand pair. More particularly, the invention relates to delivering and retaining an active agent at lymph nodes using a ligand/anti-ligand pair. Further, the invention concerns methods, compounds, compositions and kits useful for delivering and retaining an active agent at specific lymph node(s) in a mammal by administration of compositions comprising a detection or therapeutic agent and a ligand/anti-ligand pair. In a preferred embodiment, the ligand/anti-ligand pair is avidin/biotin.
  • the present invention features a ligand/anti-ligand system and colloids that are captured by draining lymph nodes when administered to a subject in vivo.
  • Ligand or anti-ligand may be conjugated to the colloid.
  • the ligand may be, for example, biotin
  • the anti-ligand may be, for example, avidin.
  • the invention features methods of delivering and retaining an active agent at targeted lymph nodes in a mammal.
  • the methods comprise the steps of (a) administering to a mammal a first composition comprising ligand conjugated to a colloid, and (b) administering to a mammal a second composition comprising anti-ligand in which the anti-ligand binds to the ligand.
  • the anti-ligand may be administered proximal to the site of the colloid-ligand conjugate administration. After the anti-ligand binds the ligand of the colloid-ligand conjugate, the aggregated colloid complex may be retained at the lymph node(s) draining the areas of interest.
  • the colloid comprises a liposome.
  • the liposome comprises one or more phospholipids.
  • the liposome comprises DSPC and/or DPPC.
  • the colloid of the invention is associated with, typically by encapsulation or binding, one or more active agents.
  • the active agents may be detection or therapeutic agents.
  • the detection or therapeutic agents are employed to detect or treat the lymph node(s) draining the area of interest.
  • the invention provides a method and composition for delivering and retaining an active agent at the sentinel lymph node(s) comprising the steps of (a) administering to a mammal a first composition comprising ligand conjugated to a colloid, and (b) administering to a mammal a second composition comprising anti-ligand in which the anti-ligand binds to the ligand.
  • the colloid may comprise an active agent, in particular, a blue dye as a detection means.
  • Anti-ligand, with or without an active agent, that binds to the ligand of the ligand-colloid-blue dye composition may be administered simultaneously or shortly after the ligand-colloid-blue dye composition is administered to a mammal.
  • the preferred administration method is by subcutaneous injection.
  • the ligand-colloid-active agent can be injected subcutaneously or intracavitary.
  • the preferred dose depends on the species and whether a therapeutic or diagnostic agent is administered. This can be administered as a single injection or in divided doses. Simultaneous with the ligand-colloid composition or after 30 minutes, more preferably at less than 15 minutes and even at less than 10 minutes, a dose of anti-ligand, with or without an active agent, may be administered subcutaneously or intracavitary.
  • the anti-ligand can be given as a single injection or in divided doses, administering the anti-ligand in two doses is preferred in certain circumstances.
  • detection of lymph nodes containing the aggregated colloid complex is accomplished using, for example, planar and single-photon emission computed tomography scans made with a gamma camera equipped with the appropriate collimator and selecting the appropriate energy windows for the detection isotope being used, such as 140 keV for Technetium-99m.
  • planar and single-photon emission computed tomography scans made with a gamma camera equipped with the appropriate collimator and selecting the appropriate energy windows for the detection isotope being used, such as 140 keV for Technetium-99m.
  • the invention may be useful in delivering and retaining one or more therapeutic agents at targeted lymph nodes.
  • the invention may also be useful in delivering and retaining one or more diagnostic agents, such as dyes or radioisotopes, at targeted lymph nodes.
  • the colloid comprises liposome
  • the ligand comprises biotin
  • the anti-ligand comprises avidin
  • the methods, compounds, compositions, and kits of the present invention are advantageous for selective detection and therapy of lymph nodes because of the significant increase in the amount of the detection and/or therapeutic agent which is available at the targeted lymph node due to the increase in retention of the detection and/or therapeutic agent at the targeted lymph node.
  • These methods, compounds, compositions, and kits are an improvement, in terms of absolute amount of detection and/or therapeutic agent retained at the lymph node, as compared to the prior art procedures which do not contemplate the use of ligand/anti-ligand systems to retain colloids in lymph nodes, thereby amplifying the amount of detection and/or therapeutic agents available at the targeted lymph node.
  • the methods of the present invention can be used to detect (either by internal procedures or by external imaging) and/or treat lymph nodes which drain specific body sites.
  • the delivery and retention of therapeutic and/or detection agents at lymph nodes using colloids such as microspheres, microcapsules, emulsions, and liposomes is contemplated.
  • the present invention greatly increases the uptake and retention of colloids in regional lymph nodes by use of a ligand/anti-ligand system, for example, the biotin/avidin system, to form complexes which are directed to and retained in the lymphatic system.
  • a ligand/anti-ligand system for example, the biotin/avidin system
  • Methods for coupling ligands to colloids, such as liposomes are known to those of skill in the art. See, for example, Schuber, in Liposomes as Tools in Basic Research and Industry , Philippot and Schuber (eds), CRC Press, Boca Raton, 21–39, 1995, incorporated herein in its entirety by reference.
  • the first lymph node encountered, or the chain of draining lymph nodes, can be targeted by the methods disclosed herein. Formation of the aggregated colloid complex prior to reaching the lymph node is important in localizing the active agent at the lymph node. When the aggregated colloid complex reaches the next encountered lymph node, it becomes retained for a prolonged time in this node.
  • the colloidal particles of the present invention are preferably in the size range 1 to 5,000 nm, more preferably 5–500 nm, and most preferably 50 to 300 nm.
  • Such systems are well transported from the site of injection and are well retained in the lymph nodes (primary and secondary). If the colloid-ligand composition is too large, it is retained at the site of injection. If the colloid-ligand composition is too small, it is transported from the site of injection into the circulation and is not retained in the lymph node(s).
  • the ligand-colloid-active agent can be administered as a single injection or in divided doses.
  • a dose of anti-ligand, with or without an active agent is administered.
  • the anti-ligand composition can be given as a single injection or in divided doses; administering the anti-ligand in two doses is preferred in certain circumstances.
  • the ligand-colloid and anti-ligand compositions are administered by subcutaneous, subdermal, submucosal, intraperitoneal, or intrapleural injection. Within one hour of the last injection, detection of lymph nodes containing the aggregated colloid complex is accomplished.
  • a radiolabeled detection agent is encapsulated in or attached to the colloid or attached to anti-ligand, detection of the lymph nodes is accomplished using for example, planar and single-photon emission computed tomography scans made with a gamma camera equipped with the appropriate collimator and selecting the appropriate energy windows for the detection isotope being used, such as 140 keV for Technetium-99m. If blue dye is encapsulated in the colloid, the lymph nodes can be visually detected.
  • the instant invention features ligand and anti-ligand compositions further comprising an imaging agent and uses for the compositions in detecting and/or monitoring tumors or sites of metastasis in a subject.
  • the ligand- or anti-ligand-imaging agent-composition is administered in vivo and monitored using a means appropriate for the label.
  • Preferred methods for detecting and/or monitoring a-ligand- or anti-ligand-imaging agent composition in vivo include Gamma Scintigraphy, Positron Emission Tomography (PET), Single Photon Emission Computer Tomography (SPECT), Magnetic Resonance Imaging (MRI), X-ray, Computer Assisted X-ray Tomography (CT), Near Infrared Spectroscopy, and Ultrasound. These techniques provide information regarding detection of neoplastic involvement, particularly of inaccessible nodes in patients with malignant diseases. Knowledge on the size of the node and the filling of nodes can also be instructive.
  • the particles so directed to the lymph nodes in detection applications will contain suitable contrast or imaging agents such as ferromagnetic materials such as iron oxide, perfluorochemicals such as perfluorooctylbromide, or gamma emitting radiolabels such as Technetium-99m, Indium-111, Gallium-67, Thallium-201, Iodine-131, 125, or 123, positron emitting radiolabels such as Fluorine-18, or those produced by neutron activation such as Samarium-153.
  • suitable contrast or imaging agents such as ferromagnetic materials such as iron oxide, perfluorochemicals such as perfluorooctylbromide, or gamma emitting radiolabels such as Technetium-99m, Indium-111, Gallium-67, Thallium-201, Iodine-131, 125, or 123, positron emitting radiolabels such as Fluorine-18, or those produced by neutron activation such as Sam
  • compositions of the present invention may be administered by standard methods, including intrapleural, intraperitoneal, subcutaneous, intraarticular, intramucosal, intramuscular, intradermal, intratumoral, interstitial, intraorgan, intracavitary, intralymphatic, intralesion, and intraosseal injection.
  • the compositions should satisfy the usual requirements for injection and are therefore administered in sterile, non-pyrogenic and preferably non-inflammatory solutions such as saline. Single or multiple injections can be used for administration of the compositions.
  • the dose administered and volume of solution injected depends on the anatomical area to be treated or investigated and will be readily determined by those of skill in the art.
  • the time required for initial localization of the colloid-ligand-anti-ligand composition in the lymph node is generally under 1 hour; however, the colloid-ligand-anti-ligand composition continues to accumulate in lymph node for 24 hours or more. Therefore, therapy or imaging can be accomplished in one short procedure.
  • localization and retention of the colloid-ligand-anti-ligand composition at specific lymph nodes limits the distribution of the colloid-ligand-anti-ligand composition in the circulation, thus the active agents of the colloid-ligand-anti-ligand composition are unlikely to produce toxic side effects at the levels required for therapy or imaging.
  • An advantage of the present invention is the flexibility of the system. For example, when a biotin-liposome-avidin complex is utilized, the complex is strongly retained in the targeted lymph node for a prolonged period, at least several days, until it is metabolized. If Technetium-99m, whose half-life is 6 hours, is employed as an active agent, it can still be imaged at least 20 hours after administration. If blue dye is employed as an active agent, lymph nodes may be visually detected at least two weeks after administration. X-ray and computerized axial tomography contrast agents may be detected for a prolonged time period, similar to that for detection of blue dye.
  • An embodiment of the present invention provides methods of increasing active agent localization and retention at a targeted lymph node of a mammalian recipient, which methods comprise:
  • a first composition comprising ligand conjugated to a colloid; and, which may contain an active agent, and ligand, for example, biotin;
  • the colloid may be associated with an active agent, such as a detection or therapeutic agent.
  • the ligand may be, for example, biotin.
  • the anti-ligand which may or may not be associated with an active agent, may be, for example, avidin.
  • the anti-ligand may be administered simultaneously or immediately after administration of the colloid-ligand composition.
  • the anti-ligand may be administered in the same location as the colloid-ligand or it may be administered at another site so that it encounters the colloid-ligand at, or just prior to reaching, the targeted lymph node.
  • Another embodiment of the present invention provides methods for detecting a sentinel lymph node of a mammal, which methods comprise:
  • a first conjugate comprising ligand conjugated to a colloid; and associated with vital blue dye and which may further contain another active agent, and ligand, for example, biotin;
  • the colloid may be associated with a detection agent, such as a radioisotope or dye.
  • the ligand may be, for example, biotin.
  • the anti-ligand which may or may not be associated with an active agent, may be, for example, avidin.
  • the anti-ligand may be administered simultaneously or immediately after administration of the colloid-ligand composition.
  • Yet another embodiment of the invention provides a composition comprising ligand conjugated to a colloid containing a radioactive label and a readily-visualized dye.
  • the active agent may be associated with the colloid or anti-ligand by any well known technique, but should be in such a way that the active agent remains associated with the colloid or anti-ligand until the point of uptake of the colloid-ligand-anti-ligand complex by the lymph nodes.
  • the association of the active agent with the colloid includes any method of incorporating the active agent into or grafting the active agent onto the colloid.
  • Imaging and Visualization Modalities of the Lymphatic System such as Gamma Scintigraphy, Positron Emission Tomography (PET), Single Photon Emission Computer Tomography (SPECT), Magnetic Resonance Imaging (MRI), X-ray, Computer Assisted X-ray Tomography (CT), Near Infrared Spectroscopy, and Ultrasound.
  • PET Positron Emission Tomography
  • SPECT Single Photon Emission Computer Tomography
  • MRI Magnetic Resonance Imaging
  • X-ray X-ray
  • CT Computer Assisted X-ray Tomography
  • Near Infrared Spectroscopy and Ultrasound.
  • the particles so directed to the lymph nodes in diagnostic applications will contain suitable contrast or imaging agents such as ferromagnetic materials such as iron oxide, perfluorochemicals such as perfluorooctylbromide, dyes, or gamma emitting radiolabels such as Technetium-99m, Indium-111, Gallium-67, Thallium-201, Iodine-123, 125, or 131, positron emitting radiolabels such as Fluorine-18.
  • suitable contrast or imaging agents such as ferromagnetic materials such as iron oxide, perfluorochemicals such as perfluorooctylbromide, dyes, or gamma emitting radiolabels such as Technetium-99m, Indium-111, Gallium-67, Thallium-201, Iodine-123, 125, or 131, positron emitting radiolabels such as Fluorine-18.
  • radionuclide labeled colloids such as Gold-198 and Yttrium-90. This includes colloids with radiosensitizers, radioprotectors, or photodynamic agents. See, for example, Coleman, Int J Radiation Onc, 42(4):781–783, 1998, incorporated herein in its entirety by reference. These could also include neutron capturing agents such as Boron-10. These agents are activated after irradiation with neutrons. See Barth et al, Cancer Res 50:1061–1071, 1990, incorporated herein in its entirety by reference.
  • Agents and diseases relevant in this regard include antigens (vaccines), DNA, RNA, peptides, biological response modifiers, antimicrobial agents for treatment of infection of the nodes such as in filariasis, brucellosis, tuberculosis, HIV, and antitumour agents such as mitomycin C, bleomycin, etc.
  • compositions of the present invention may be administered by standard methods and particularly by subcutaneous or intracavitary injection.
  • the compositions should satisfy the usual requirements for injection and are therefore administered in sterile, non-pyrogenic solutions such as saline.
  • Single or multiple injections can be used for administration of the compositions.
  • the dose administered and the volume of solution injected depends on the anatomical area to be treated or investigated.
  • Colloidal particles can have an important role in characterizing the properties of the lymphatic system as well as a possible role in delivering active agents to the lymphatic system.
  • a wide range of materials has been examined to include solid particles, emulsions and vesicles (liposomes).
  • the distribution of colloidal agents depends strongly on their particle size; for example, colloids suggested for lymphoscintigraphy were found to have a median size of about 40–60 nm. Uptake into regional lymph nodes after, for example, subcutaneous administration is generally quite small.
  • colloids that will be useful in the present invention.
  • Colloidal particles suitable for use in the present invention include microspheres and nanoparticles, starburst dendrimers (see Wilbur et al., Bioconjug Chem 9:813–825, 1998, incorporated herein in its entirety by reference), microcapsules or nanocapsules, emulsions, microemulsions, liposomes and mimics of lipoproteins and chylomicrons.
  • Suitable materials for producing these include polylactic acid and polyglycolic acid and their mixtures, polyactidecoglycolide mixtures, polymalic acid, polyalkylcyanoacrylates, polyanhydrides, polycaprolactones, polyphosphazenes, natural materials such as hyaluronic acid, albumin, dextran, gelatin, starch, collagen, polysaccharides and derivatives thereof and vegetable oils such as soybean oil. Particular success has been achieved with semi-synthetic phospholipids and cholesterol.
  • solutions containing the colloids of the present invention will be administered in a manner compatible with the dosage formulation and in such amount as is effective for detection and therapeutic purposes.
  • the formulations are administered in the form of injectable solutions.
  • aqueous solutions For subcutaneous or intracavitary administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for interstitial, subcutaneous, and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the lymph node(s) targeted. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.
  • the active agent may be incorporated in a liposome or conjugated to the surface of the liposome.
  • Liposomes are lipid bilayer structures that may be formed on addition of an aqueous solution to lipids. Liposomes can and do take on a variety of shapes and sizes, both spherical and non-spherical, including but not limited to distorted, flattened or “collapsed” shapes, and even broken or fragmented shapes. They may be vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes may have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap aqueous solution between the lipid bilayers.
  • Liposomes have been described as potential agents for targeting delivery of diagnostic or therapeutic agents to a wide range of organ systems and diseases. Such targeting is due primarily to a physical feature of the liposome, such as size, charge, and lipid composition, and is not due to specific site-directed targeting. Phillips et al., in Handbook of Targeted Delivery of Imaging Agents , CRC Press, 149–173, 1995, incorporated herein in its entirety by reference.
  • Suitable liposome systems can be prepared with a mixture of phospholipids and cholesterol. The molar ratio can be varied for the desired composition.
  • Polyethyleneglycol-phosphatidylethanolamine is an example of a modified phospholipid that can be used to provide liposomes with appropriate surface characteristics.
  • liposomes The preparation of liposomes is well described in the literature (see, for example, Litzinger et al., Biochim Biophys Acta. 1127(3):249–254, 1992; New, in Liposomes: A Practical Approach , New (ed), Oxford University Press, NY, 33–104, 1990).
  • the mixture of phospholipid and cholesterol is dissolved in chloroform, placed in a round bottom glass tube, and the organic solvent is evaporated under vacuum.
  • the lipid film so obtained is suspended by the addition of buffer to form multilamellar liposomes. Sonication, homogenization, microfluidization, dialysis, or passage of these liposomes through nucleopore filters results in the formation of small unilameller vesicles.
  • the materials which may be utilized in preparing liposomes for use in the present invention include any of the materials or combinations thereof known to those skilled in the art as suitable for liposome preparation.
  • the lipids used may be of either natural or synthetic origin. The particular lipids are chosen to optimize the desired properties.
  • Lipids which may be used to create liposome microspheres include but are not limited to: lipids such as fatty acids, lysolipids, phosphatidylcholine with both saturated and unsaturated lipids including dioleoylphosphatidylcholine; dimyristoylphosphatidyl-choline; dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoyl-phosphatidylcholine; distearoylphosphatidylcholine; phosphatidylethanolamines such as dioleoylphosphatidylethanolamine; phosphatidylserine; phosphatidylglycerol; phospha-tidylinositol, sphingolipids such as sphingomyelin; glycolipids such as ganglioside GM1 and GM2; glucolipids; sulfatides; glycos
  • cationic lipids such as DOTMA, N-1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride; DOTAP, 1,2-dioleoyloxy-3-(trimethylammonio)propane; and DOTB, 1,2-dioleoyl-3-(4′-trimethyl-ammonio)butanoyl -sn-glycerol may be used.
  • DOTMA N-1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride
  • DOTAP 1,2-dioleoyloxy-3-(trimethylammonio)propane
  • DOTB 1,2-dioleoyl-3-(4′-trimethyl-ammonio)butanoyl -sn-glycerol
  • the molar ratio of cationic lipid to non-cationic lipid in the liposome may be, for example, 1:1000, 1:100, preferably, between 2:1 to 1:10, more preferably in the range between 1:1 to 1:2.5 and most preferably 1:1 (ratio of mole amount cationic lipid to mole amount non-cationic lipid, e.g., DPPC).
  • a wide variety of lipids may comprise the non-cationic lipid when cationic lipid is used to construct the microsphere.
  • this non-cationic lipid is dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine or dioleoylphosphatidylethanolamine.
  • lipids bearing cationic polymers such as polylysine or polyarginine may also be used to construct the microspheres and afford binding of a negatively charged therapeutic, such as genetic material, to the outside of the microspheres.
  • carbohydrate-bearing lipids may be employed for in vivo targeting, as described in U.S. Pat. No. 4,310,505, the disclosures of which is hereby incorporated herein by reference, in its entirety.
  • the most preferred lipids are phospholipids, preferably DPPC and DSPC, and most preferably DSPC. It is also preferable to include cholesterol in the lipid formulation.
  • the size of active agent-containing liposomes can be adjusted, if desired, by a variety of procedures including extrusion, filtration, sonication, homogenization, employing a laminar stream of a core of liquid introduced into an immiscible sheath of liquid, extrusion under pressure through pores of defined size, and similar methods, in order to modulate resultant liposomal biodistribution and clearance.
  • the ability to target specific lymph nodes for detection, diagnosis and/or therapy is highly desirable.
  • the biotin/avidin system can be used in methods for targeting lymph nodes.
  • Avidin is a glycoprotein, found in egg whites, that has an extremely high binding affinity for biotin, a natural B-complex vitamin found in every living cell.
  • Streptavidin derived from Streptomyces avidinii , is similar to avidin in its chemical and physical characteristics and identical to avidin in its ability to bind biotin. Both avidin and streptavidin have a tetravalency for biotin. Streptavidin can be used in place of avidin in many applications because of its low nonspecific tissue binding property.
  • Both avidin and streptavidin have a tetravalency for biotin, thus permitting amplification when the former bind to biotin.
  • the present invention encompasses natural avidin and its derivatives and analogs capable of being bound by biotin, in particular, avidin modified to have lower immunogenicity.
  • Natural biotin is a water-soluble vitamin found in every living cell.
  • the present invention encompasses natural biotin and its derivatives and analogs capable of being bound by avidin.
  • Exemplary biotin molecules include 2′-thiobiotin; 2′-iminobiotin; 1′-N-methoxycarbonylbiotin; 3′-N-methoxycarbonylbiotin; 1-oxybiotin; 1-oxy-2′-thiobiotin; 1-oxy-2′-iminobiotin; 1-sulfoxidebiotin; 1-sulfoxide-2′-thiobiotin; 1-sulfoxide-2′-iminobiotin; 1-sulfonebiotin; 1-sulfone-2′-thiobiotin; 1-sulfone-2′-iminobiot imidazolidone derivatives such as desthiobiotin (d and dl optical isomers), dl-desthiobiotin methyl ester, dl-desthiobio
  • biotin molecules for use in the practice of the present invention are natural biotin conjugated to the headgroup region of a phospholipid. These exemplary biotin molecules may be produced substantially in accordance with known procedures therefore. Conjugation of the exemplary biotin molecules to colloids proceeds substantially in accordance with known procedures therefore and with procedures described herein with regard to biotin conjugation.
  • ligand/anti-ligand systems useful in the present invention include synthetically designed ligand/anti-ligand pairs with high affinity based on polyvalency.
  • Rao et al. described one pair with higher affinity than biotin/avidin using tris vancomycin carboxamide for binding to the trivalent ligand derived from D-Ala-D-Ala (Rao et al., Science, 280(5364):708–711, 1999, incorporated herein in its entirety by reference).
  • Another substitution includes antibody/antigen pairs.
  • the biotin ligand on the surface of the colloid could be replaced by an antigen.
  • an aggregate complex could form.
  • the antibody could serve as the ligand and be associated with the surface of the colloid while the corresponding antigen could serve as the anti-ligand (Leserman et al., in Liposomes From Biophysics to Therapeutics, Ostro (ed), Marcel Dekker, New York, pp.
  • lectin/complementary carbohydrate Another example of an alternate ligand/anti-ligand pair is lectin/complementary carbohydrate.
  • lectin could serve as the ligand.
  • Lectins on the surface of a colloid could aggregate after association with the complementary carbohydrate anti-ligand (Leserman et al., in Liposomes From Biophysics to Therapeutics , Ostro (ed) Marcel Dekker, New York, pp. 157–194, 1987; and Hermanson, Bioconjugate Techniques , Academic Press, San Diego, Calif., pp. 548–549, 1996, incorporated in their entirety by reference).
  • the lectin could serve as the anti-ligand.
  • ligand/anti-ligand pairs such as the folate/folate receptor, fibrin/plasminogen, and sialyl Lewis X/E-selectin could potentially replace biotin/avidin as the ligand/anti-ligand pair (Gabizon et al., Bioconjugate Chem 10:289–298, 1999; Heeremans et al., Thromb Haemost 75(1):134–139, 1996; DeFrees et al., J Am Chem Soc 118:6101–6104, 1996, all of which are incorporated in their entirety by reference).
  • Kits comprising (1) ligand conjugated to a colloid containing an active agent, and (2) anti-ligand with or without an active agent comprise another aspect of the present invention.
  • the detection/therapeutic kits comprising the active agents disclosed herein will generally contain, in suitable container means, a detection- or therapeutically-effective amount of an active agent.
  • the kit may have a single container means that contains the ligand-targeting colloid-active agent, or it may have distinct container means for each compound.
  • the components of the kit may be provided as liquid solution(s), or as dried powder(s).
  • the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
  • the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • the ligand-colloid-active agent composition and the anti-ligand-active agent composition may also be formulated into syringeable compositions.
  • the container means may itself be a syringe, or other such like apparatus, from which the formulation may be administered into the body, preferably by injection or even mixed with the other components of the kit prior to injection. Dosage of each of the compositions will vary from subject to subject depending upon the use of the compositions, size of the subject, potential location of the lymph node to be targeted, body weight of the subject, etc. The calculation and adjustment of dosages of a variety of compositions is well-known to those of skill in the art.
  • components of the kit may be provided as dried powder(s).
  • the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
  • the container means will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the ligand-colloid-active agent compositions may be placed, preferably, suitably allocated.
  • the kit will also generally contain a second vial or other container into which the anti-ligand-active agent compositions may be placed.
  • the kits may also comprise a second/third container means for containing a sterile, pharmaceutically acceptable buffer or other diluent.
  • kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.
  • a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.
  • the vials may be prepared in such a way as to permit direct introduction of the composition into an intravenous drug delivery system.
  • kits of the invention may also comprise, or be packaged with, an instrument for assisting with the injection/administration or placement of the compositions of the invention within the body of a mammal.
  • an instrument may be a syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle.
  • the detection/therapeutic agents used in the methods of the present invention can be any or multiples of the following:
  • A—diagnostic or therapeutic agents e.g., alpha-, beta-, gamma-, positron-, x-ray- and fluorescence-emitters; electron- and neutron-capturing agents, paramagnetic and ferromagnetic agents);
  • C cytotoxic agents (e.g., drugs, toxins, hormones, cytokines, hormone antagonists, receptor antagonists);
  • D biological response modifier agents (e.g., vitamins, cytokines, autocrines, peptides, anti-angiogenesis agents, certain hormones and drugs);
  • gamma-emitters positron-emitters, x-ray emitter, paramagnetic or ferromagnetic ions, and fluorescence-emitters are suitable for detection and/or therapy, while beta- and alpha-emitters and neutron-capturing agents can be used for therapy.
  • Therapeutic Agents are suitable for detection and/or therapy, while beta- and alpha-emitters and neutron-capturing agents can be used for therapy.
  • any of a variety of therapeutics may be encapsulated in the colloids or conjugated to the surface of the colloids.
  • Many pharmaceutical compositions are known which have cytotoxic effects on cells. They are to be found in compendia of drugs, such as the Merck Index, Goodman and Gilman, and the like, and in the references cited above. Any such pharmaceutical composition can be conjugated to anti-ligand or loaded into the ligand-colloid by conventional means well known in the art.
  • Preferred therapeutic agents suitable for use herein include conventional chemotberapeutics, such as vinblastine, doxorubicin, bleomycin, methotrexate, 5-fluorouracil, 6-thioguanine, cytarabine, cyclophosphamide and cisplatinum, as well as other conventional chemotherapeutics as described in DeVita et al., Cancer: Principles and Practice of Oncology, 5 th ed ., J. B. Lippincott Co., Philadelphia, Pa., Chapter 19, 375–512, 1997, incorporated herein by reference.
  • the preferred therapeutic agent for use in the present invention will depend on the particular tumor or type of lesion to be treated.
  • cytotoxic agents useful in the present invention are listed in Goodman et al., The Pharmacological Basis of Therapeutics, 6 th Ed ., Gilman et al., (eds.), Macmillan Publishing Co., New York, 1980, incorporated herein in its entirety by reference.
  • taxol include taxol; nitrogen mustards, such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard and chlorambucil; ethylenimine derivatives, such as thiotepa; alkyl sulfonates, such as busulfan; nitrosoureas, such as carmustine, lomustine, semustine and streptozocin; triazenes, such as dacarbazine; folic acid analogs, such as methotrexate; pyrimidine analogs, such as fluorouracil, cytarabine and azaribine; purine analogs, such as mercaptopurine and thioguanine; vinca alkaloids, such as vinblastine and vincristine; antibiotics, such as dactinomycin, daunorubicin, doxorubicin, bleomycin, mithramycin and mitomycin; enzymes, such as L-a
  • hormone suppressants useful in the present invention are listed in Goodman et al, The Pharmacological Basis of Therapeutics, 6 th Ed , Gilman et al. (eds), Macmillan Publishing Co. New York, 1980. These include adrenocortical suppressants, such as mitotane; hormones and antagonists, such as adrenocortisteroids (prednisone), progestins (hydroxyprogesterone caproate, medroprogesterone acetate and megestrol acetate), estrogens (diethylstilbestrol and ethinyl estradiol), anti-estrogens (tamoxifen), and androgens (testosterone propionate and fluoxymesterone).
  • adrenocortical suppressants such as mitotane
  • hormones and antagonists such as adrenocortisteroids (prednisone), progestins (hydroxyprogesterone caproate, medroprogesterone
  • Drugs that interfere with intracellular protein synthesis can also be used in the methods of the present invention; such drugs are known to those skilled in the art and include puromycin, cycloheximide, and ribonuclease.
  • more than one therapeutic may be delivered using the colloid.
  • a single colloid may contain more than one therapeutic or colloid containing different therapeutics may be co-administered.
  • a therapeutic radionuclide such as Yttrium-90 and chemotherapeutic agent such as vincristine may be administered at the same time.
  • prodrugs may be encapsulated in the colloids, and are included within the ambit of the term therapeutic, as used herein.
  • Prodrugs are well known in the art and include inactive drug precursors which, when exposed to high temperature, metabolizing enzymes, cavitation and/or pressure, in the presence of oxygen or otherwise, or when released from the colloid, will form active drugs.
  • Such prodrugs can be activated in the method of the invention, upon the application of ultrasound to the prodrug-containing colloid with the resultant cavitation, heating, pressure, and/or release from the colloid.
  • Suitable prodrugs will be apparent to those skilled in the art, and are described, for example, in Sinkula et al., J Pharm Sci 64:181–210, 1975, the disclosure of which is hereby incorporated herein by reference in its entirety.
  • Radioisotopes may also be used as therapeutic agents. Any conventional method of radiolabeling which is suitable for labeling isotopes for in vivo use will be generally suitable for labeling therapeutic agents according to the present invention.
  • Isotopes preferred for therapeutic use include: Actinium-225, Bismuth-212, Lead-212, Bismuth-213, Iodine-125, Iodine-131, Rhenium-186, Rhenium-188, Silver-111, Platinum-197, Palladium-109, Copper-67, Phosphorus-32, Phosphorus-33, Yttrium-90, Scandium-47, Samarium-153, Lutetium-177, Rhodium-105, Praseodymium-142, Praseodymium-143, Terbium-161, Holmium-166, and Gold-199.
  • Detection agents of use in the present invention include radioisotopes and dyes. Any conventional method of radiolabeling which is suitable for labeling isotopes for in vivo use will be generally suitable for labeling detection agents according to the present invention.
  • Internal detection procedures include intraoperative, intravascular or endoscopic, including laproscopic, techniques, both surgically invasive and noninvasive.
  • Suitable radioisotopes for the methods of the present invention include: Actinium-225, Astatine-211, Iodine-123, Iodine-125, Iodine-126, Iodine-131, Iodine-133, Bismuth-212, Bromine-77, Indium-111, Indium-113m, Gallium-67, Gallium-68, Ruthenium-95, Ruthenium-97, Ruthenium-103, Ruthenium-105, Mercury-107, Mercury-203, Rhenium-186, Rhenium-188, Tellurium-121m, Tellurium-122m, Tellurium-125m, Thulium-165, Thulium-167, Thulium-168, Technetium-99m, Fluorine-18, Silver-111, Platinum-197, Palladium-109, Copper-67, Phosphorus-32, Phosphorus-33, Yttrium-90, Scandium-47, Samarium-153, Lutetium-177, Rhodium-105
  • the most preferred radioisotope for use in the current invention is Technetium-99m.
  • the radioisotope will emit a particle or ray in the 10–7,000 keV range, more preferably in the 50–1,500 keV range, and most preferably in the 80–250 keV range.
  • Isotopes preferred for external imaging include: Iodine-123, Iodine-131, Indium-111, Gallium-67, Ruthenium-97, Technetium-99m, Cobalt-57, Cobalt-58, Chromium-51, Iron-59, Selenium-75, Thallium-201, and Ytterbium-169.
  • Technetium-99m is the most preferred radioisotope for external imaging in the present invention.
  • Isotopes most preferred for internal detection include: Iodine-125, Iodine-123, Iodine-131, Indium-111, Technetium-99m and Gallium-67. Technetium-99m is the most preferred isotope for internal detection.
  • Dyes may also be used as detection agents.
  • blue dye In order to aid with localization of the sentinel lymph node for a tumor, blue dye has been injected around the tumor. The blue dye travels into the lymphatic system and through the sentinel lymph node. While this permits visualization of lymph nodes, for example, during surgical procedures, it does not permit detection of the sentinel lymph node because the blue dye does not localize at the first lymph node encountered, but passes on to lymph nodes further along the lymph node chain.
  • Incorporation of blue dye with the colloid of the aggregated colloid complex of the present invention provides for retention of the blue dye at the first lymph node encountered, or in the chain of draining lymph nodes, depending upon the timing of administration of the anti-ligand-active agent.
  • Liposomes were comprised of distearoyl phosphatidylcholine (DSPC) (Avanti) Polar Lipids, Pelham, Ala.); cholesterol (Chol) (Calbiochem, San Diego, Calif.); Nbiotinoyl distearoyl phosphoethanolamine (Biotin-DSPE) (Northern Lipids, Vancouver, Canada); and ⁇ -tocopherol (Aldrich, Milwaukee, Wis.). All lipids were used without further purification. The lipids were mixed in chloroform at a total molar ratio of 58:39:1:2 (DSPC:Chol:Biotin-DSPE: ⁇ -tocopherol).
  • DSPC distearoyl phosphatidylcholine
  • cholesterol Chol
  • Biotin-DSPE Nbiotinoyl distearoyl phosphoethanolamine
  • ⁇ -tocopherol Aldrich, Milwaukee, Wis.
  • Chloroform was then removed by rotary evaporation to form a lipid film.
  • the lipid film was stored overnight in a vacuum desiccator to remove organic solvent.
  • Samples were rehydrated with 300 mM sucrose (Sigma, St. Louis, Mo.) in sterile water for injection and warmed to 55° C. for 15 min with periodic vortexing until all of the lipids were in suspension.
  • the resultant multilamellar vesicles formed from rehydration were then frozen in liquid nitrogen and lyophilized.
  • the resultant dry sugar-lipid preparations were then rehydrated with 200 mM reduced glutathione (GSH) (Sigma, St.
  • the diluted lipid suspensions were then extruded through a series (2 passes, 2 ⁇ ; 2 passes, 400 nm; 5 passes, 100 nm) of polycarbonate filters (Lipex Extruder, Vancouver, Canada) at 55° C.
  • the extruded lipid solution was then washed in Dulbecco's phosphate buffered saline containing 75 mM sucrose and centrifuged at 200,000 ⁇ g for 45 min to remove unencapsulated GSH and sucrose, and to concentrate the liposome sample. The washing step was repeated 3 times.
  • the final liposome pellet was resuspended in Dulbecco's phosphate buffered saline pH 6.3 containing 300 mM sucrose at a lipid concentration of 102 mM and stored in the refrigerator at 4° C.
  • Liposomes encapsulating blue dye were comprised of the same lipids as for the biotin-liposomes.
  • the liposomes were processed in an identical fashion as listed above except patent blue violet dye (Sigma, St Louis, Mo.; CI 42045) was included during processing in the following manner: 1).
  • the dry sugar-lipid preparation was rehydrated with Dulbeccco's phosphate buffered saline pH 6.3 containing 200 mM GSH and 10 mg/ml of blue dye at a lipid concentration of 102 mM.
  • the liposome solution was diluted at a volume/volume ratio of 1 part lipid suspension to 2 parts Dulbecco's phosphate buffered saline containing 100 mM GSH, 150 mM sucrose and 10 mg/ml of blue dye.
  • the Technetium-99m ( 99m Tc) carrier found most preferable is an alkylenepropyleneamine oxime that complexes with 99m Tc and can be purchased as a lyophilized preparation (CeretecTM, Nycomed-Amersham, Arlington Hgts, Ill.).
  • HMPAO is mixed with sterile eluate from a 99m Tc-generator
  • the generator eluate may be adjusted to a radioactive concentration of between 037–1.11 GBq (10–30 mCi) in 5 ml by dilution with preservative free, nonbacteriostatic saline prior to mixing with 0.5 mg of HMPAO.
  • the 99m Tc complex forms almost immediately and is incubated for 5 min at room temperature.
  • This mixture of 99m Tc-HMPAO (0.5 ml, 1 mCi) was then incubated with 1 ml (102 mM of lipid) of either biotin-liposomes containing GSH alone or biotin-liposomes coencapsulating GSH and blue dye prepared as described above.
  • the 99m Tc-HMPAO-liposome mixture was incubated for 15–30 min at room temperature with intermittent swirling.
  • the radiolabeled liposomes were then separated from any free 99m Tc by passage over a Sephadex G-25 column (PD10 column, Pharmacia Biotech, Uppsala, Sweden) equilibrated with Dulbecco's phosphate buffered saline pH 6.3. Labeling efficiencies were checked by determining the activity before and after column separation of the 99m Tc-biotin-liposomes using a dose calibrator (Radix, Houston, Tex.). Labeling efficiencies averaged 97% for biotin-liposomes containing GSH and 92% for biotin-liposomes coencapsulating GSH and blue dye. Postcolumn preparations of the 99m Tc-biotin-liposomes were used immediately for injection.
  • avidin-active agents for therapy include a) the chemotherapeutic agent cisdiamminedichloroplatinum (II) complexed to a carboxymethyl dextran-avidin conjugate; and b) boron-10 chlorpromazine conjugated to avidin for use in boron neutron capture therapy (Schechter et al., In J Cancer 48(2):167–172, 1991 and Komura et al., Melanoma Research 1:397–403, 1992, incorporated in their entirety by reference).
  • chemotherapeutic agent cisdiamminedichloroplatinum (II) complexed to a carboxymethyl dextran-avidin conjugate
  • boron-10 chlorpromazine conjugated to avidin for use in boron neutron capture therapy Schoechter et al., In J Cancer 48(2):167–172, 1991 and Komura et al., Melanoma Research 1:397–403, 1992, incorporated in their entirety by reference.
  • avidin-active agents include using a monoclonal antibody-avidin conjugate to increase delivery of therapeutic agents through the blood brain barrier; and b) delivery of a luteinizing hormone releasing hormone-avidin conjugate for improved antibody titer to luteinizing hormone releasing hormone for disruption of reproductive function (Kang et al., J Pharm Exptl Ther 269: 344–350, 1994 and Tiong et al., Vaccine 11:425–430, 1993, incorporated in their entirety by reference).
  • the active agent is a radioisotope for detection or therapy
  • the preferred method of preparation will depend, for example, on the isotope chemistry.
  • Methods for conjugating anti-ligand, for example, avidin, to a detection or therapeutic agent include the following: (a) the chloramine-T or Bolton-Hunter procedures for conjugating iodine, (b) the procedures described by Griffiths et al., Cancer Res 51(17):4594–4602, 1991, or Fritzberg et al., U.S. Pat. No.
  • An application for the method of the present invention is for gamma scintigraphy.
  • a suitably radiolabeled conjugate is administered with the intention of obtaining an image of the lesion.
  • the method of the invention can be practiced either with scintigraphic or magnetic resonance imaging agents.
  • a combination of these imaging agents can also be used, although this requires more complex instrumentation and data processing.
  • Scintigraphic imaging according to the method of the invention is effected by obtaining a scintigram of the lesion of interest.
  • the scintigram is normally taken by a gamma imaging camera having one or more windows for detection of energies in the 50–600 keV range.
  • Use of radioisotopes with higher energy, beta, or positron emissions would entail use of imaging cameras with the appropriate detectors, all of which are conventional in the art.
  • the scintigraphic data can be stored in a computer for later processing.
  • Magnetic resonance imaging is effected in an analogous manner to scintigraphic imaging except that the imaging agents will contain magnetic resonance (MR) enhancing species rather than radioisotopes.
  • MR magnetic resonance
  • the magnetic resonance phenomenon operates on a different principle from scintigraphy. Normally, the signal generated is correlated with the relaxation times of the magnetic moments of protons in the nuclei of the hydrogen atoms of water molecules in the region to be imaged.
  • the magnetic resonance image enhancing agent acts by increasing the rate of relaxation, thereby increasing the contrast between water molecules in the region where the imaging agent accretes and water molecules elsewhere in the body.
  • the effect of the agent is to decrease both T 1 and T 2 , the former resulting in greater contrast while the latter results in lesser contrast.
  • the phenomenon is concentration-dependent, and there is normally an optimum concentration of a paramagnetic species for maximum efficacy.
  • This optimal concentration will vary with the particular agent used, the locus of imaging, the mode of imaging, i.e., spin-echo, saturation-recovery, inversion-recovery and/or various other strongly T 1 -dependent or T 2 -dependent imaging techniques, and the composition of the medium in which the agent is dissolved or suspended. These factors, and their relative importance are known in the art.
  • the MR image enhancing agent must be present in sufficient amounts to enable detection by an external camera, using magnetic field strengths which are reasonably attainable and compatible with patient safety and instrumental design.
  • the requirements for such agents are well known in the art.
  • MRI contrast agents are well known in the art and include, for example, Gadolinium, Iron, Manganese, Rhenium, Europium, Lanthanium, Holmium, and Terbium.
  • the MR scans are stored in a computer and the images processed analogously to the scintigraphic data.
  • CT imaging allows for 3 dimensional pictures by backprojection reconstruction of images obtain in radial fashion around a patient
  • Contrast for both plain X-ray and CT imaging is effected in a manner analogous to that described for scintigraphic and MRI imaging except that the imaging agents provide contrast by increasing the attenuation of the X-ray beam. Attenuation of the X-ray beam depends on the energy of the beam and the tissue mass attenuation coefficient. The greater the average proton number Z of the tissue, the greater the attenuation.
  • CT and X-ray contrast imaging agents are therefore based on high Z atoms such as iodine.
  • ultrasound as a diagnostic imaging modality has increased in recent years.
  • Ultrasound imaging can be enhanced by the use of ultrasound imaging agents.
  • the agents increase contrast by reflecting and scattering ultrasound waves.
  • the use of ultrasound contrast can increase delineation of important structures in the body and increase diagnostic ability.
  • Much recent effort has been dedicated to the targeting of ultrasound contrast colloidal to tissues of interest in the body.
  • Common ultrasound contrast colloids include air microbubbles coated with protein and liposomes which encapsulate air. Factors affecting the quality of ultrasound contrast are well known. See Klibanov, Adv Drug Deliv Rev, 37:139–157, 1999, incorporated in its entirety by reference.
  • Neutron capture therapy consists of administration of a nonradioactive isotope which can be split into a heavy ion and an alpha particle upon irradiation with thermal neutrons.
  • the advantage of this system is that the therapeutic radiation can be better controlled and only the site irradiated with the thermal neutrons and containing the neutron capture agent will receive a high dose of radiation.
  • the most commonly used isotope for this therapy is boron-10.
  • the delivery of boron-10 to specific sites in the body using colloids has been under development.
  • One proposed system uses boron-10 encapsulated in a liposome.
  • lymph node delivery system with BCNT would have advantages to one skilled in the art because the site of initial subcutaneous injection would not receive any radiation while the site of uptake in the lymph node could receive the targeted radiation therapy.
  • Vaccine adjuvants are agents when given in combination with an antigen, greatly increase the immune response to the antigen.
  • Vaccine adjuvants are essentially antigen delivery systems, however, the mechanisms and locations involved in the delivery of the antigen are poorly understood.
  • Most common adjuvants are colloids. Typical colloids used are aluminum hydroxide colloids and liposome colloids. (See Theory and Practical Application of Adjuvants . Stewart-Tull (ed), John Wiley & Sons Ltd., Chichester, England, 1995, incorporated herein by reference.) The most recent information indicates that antigen delivery to the lymph node and induction of lymph node hypercellularity are important aspects of adjuvant function.
  • leg raising was initialized for 1 min at 30, 40, 50 and 60 min after liposome injection.
  • the avidin was injected into the right foot while the left foot served as a control.
  • the avidin was injected into both feet of the same rabbit in 4 rabbits and 4 control rabbits were administered no avidin.
  • biotin-liposomes encapsulating only glutathione and labeled using the 99m Tc-HMPAO technique were studied.
  • Four rabbits were subcutaneously injected in the foot pads of both feet with 99m Tc-biotin-liposomes in 0.3 ml volume. This is an asymmetric study, since only one foot was then subcutaneously injected with 5 mg of avidin in 0.3 ml of saline 5 min after injection of the 99m Tc-biotin-liposomes.
  • the avidin was subcutaneously injected on the right rabbit foot 2 cm proximal from the liposome injection.
  • Scintigraphic imaging with a gamma camera was performed of the rabbits for the first hour after liposome injection and then at 20 hours to follow the movement of the 99m Tc-biotin-liposomes in relationship to the avidin. These images are analyzed by drawing a region of interest around the site of the liposome injection and around the popliteal lymph nodes.
  • the rabbits were sacrificed at 20 hr and tissue samples were taken of the popliteal nodes and counted in a scintillation well counter. The percent uptake of 99m Tc-biotin-liposomes in the nodes was compared to a standard representing the total amount injected in each foot pad.
  • the iliac nodes also had increased retention in avidin rabbits compared with control rabbits.
  • the avidin also greatly reduced the uptake of 99m Tc-biotin-liposomes by the liver and spleen.
  • Biotin-liposomes labeled with 99m Tc can encapsulate blue dye for visually marking the first lymph node encountered.
  • the blue dye can also be considered as an example of drug delivery to a lymph node.
  • Other drugs such as anticancer agents, antiviral agents, vaccines, photodynamic dyes, antibiotics, and therapeutic radionuclides, could also be delivered instead of blue dye.
  • the most remarkable finding was the visual appearance of the popliteal node on the side that received the avidin. It was very blue at 20 hours after liposome injection. The blue staining of the lymph node allows it to be readily identified during surgery. The long retention of the blue liposomes in the lymph node has great potential advantages for the variety of protocols which will allow easy recognition of the sentinel lymph node.
  • the liposomes can be injected the day before surgery and will still be visualized the following day during surgery.
  • Results are shown in Table 4. Imaging studies show that there was a statistically significant increase in uptake even after the 3rd injection. There was 4.8% in the side injected with avidin versus 1.0% in the control side Rabbits were imaged at baseline, 2 wk and 4 wk.
  • Results are shown in Table 6.
  • the experimental group had a completely different distribution compared to the control group.
  • the control group had an expected distribution of liposomes in the blood, spleen and liver while the experimental group had minimal activity in these organs.
  • the experimental group had significantly elevated lymph node uptake by lymph nodes in the abdomen and the mediastinal regions.
  • the experimental animal had approximately 8 times more accumulation in the hepatic node compared to the control animal.
  • the biodistribution results are shown in Table 7.
  • a second node was visualized in the abdominal region of the experimental animal that was not visualized in the control animal. Differences between the control and experimental animals were clearly visualized.
  • lymph nodes draining a region of the colon can be targeted using identification of the cancer with colonoscopic visualization and subsequent injection of the submucosa near the cancer with 99m Tc-blue-biotin liposomes and avidin. This could greatly increase the delivery of therapeutic agents such as drugs and therapeutic radionuclides to the lymph nodes that drain the region of the colon carcinoma.
  • compositions, compounds, methods, and kits of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions, compounds, methods, and kits, and in the steps of the methods described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

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